Host-vector system which can be used in gene therapy

ABSTRACT

The invention relates to a system for expressing a transgene in a target cell or a human or animal cell, characterized in that it consists of a eukaryotic cell established as a line, into which there have been transfected:  
     a) a recombinant viral sequence in which a gene has been deleted totally or partially and substituted by the said transgene at the level of this gene;  
     b) a nucleic acid sequence including a sequence encoding the deleted protein, which sequence is in dependence on a promoter and is combined, where appropriate, with the said transgene, and flanked at its 3′ end a polyadenylation site;  
     the said recombinant viral genome and the said sequence, carried by one or two plasmid supports, being capable of trans-complementing each other and allowing the said host cell to produce defective infectious viruses.

[0001] The present invention relates to a new approach for gene therapyapplied especially to the elimination of certain categories of cells,such as tumor cells or certain cells infected by viruses, by theproduction of recombinant retroviruses from new genetic constructs.

[0002] The concept of gene therapy by killer genes or suicide genes hasbeen developed since 1986 by numerous approaches. It involves theexpression of a gene, this expression allowing the conversion of anontoxic substance or a toxic substance by the cell. This concept ofgene therapy is applicable and transposable to any gene whose expressionin a target cell makes it possible to convert an inactive substance toan active substance, or vice versa, leading either to a destruction(example of the suicide gene), or to a restoration of certain functions.

[0003] In all that follows, the application of these concepts to suicidegenes is more particularly developed given the accessibility of itsexperimental use. The transposition of the means of the invention to anytype of transgene whose expression leads to the effect of a substancebeing modified in the direction of an activation or inactivation isnevertheless within the capability of persons skilled in the art.

[0004] Herpes Simplex virus type I thymidine kinase (ESV1-TK) is theenzyme which has been the subject of the highest number ofinvestigations relating to suicide genes.

[0005] This enzyme, which is atoxic for eukaryotic cells, has thecharacteristic of being able to convert certain nucleoside analogs suchas aciclovir (ACV) or ganciclovir (GCV) to monophosphate-containingmolecules, of which cellular kinases are normally incapable (G. B.Elion, J. Antimicrob. Chemother. 12:9-17 (1983)). These nucleosidemonophosphates are then converted by cellular enzymes to nucleotidetriphosphates which can be used during the synthesis of DNA and blockthe elongation process thereby causing the death of the cell. Thenucleoside triphosphate analog is therefore only toxic for dividingcells.

[0006] All the advantages which can be gained from the use of this typeof conditional toxins can therefore be understood when applied to gene,especially anticancer, therapy.

[0007] Firstly, only a conditional toxicity makes it possible togenerate stable cell clones producing the pseudo-viral particles capableof producing such a transgene and transferring the suicide gene into thetarget cells. Indeed, these cells simply have to be cultured in theabsence of ACV or GCV since HSV1 thymidine kinase is not toxic for thecell in the absence of these drugs.

[0008] Next, in the event of a side effect of the treatment, stoppingthe administration of ACV or GCV causes the toxicity due to thetransgene to cease immediately; in addition, adjustment of the doses ofthe nucleoside analog makes it possible to selectively destroy the cellsstrongly expressing the transgene while preserving the cells in whichthe gene is weakly expressed; this toxicity which is restricted todividing cells is a great advantage especially for the treatment ofcancer cells.

[0009] Finally, experimental data in vitro and in vivo have shown thatcells not expressing HSV1-TK, but which are in contact with the latter,were also destroyed by the treatment with ACV (“Metabolic Cooperation”or “bystander effect”) (Moolten F. L., 1986, Cancer Research,46:5276-5281 and Culver K. W. et al., 1992, Science 256:1550-1552).

[0010] The mechanism of this effect is still not understood, but it ispossible that the nucleoside triphosphate analogs can pass from one cellto another via “gap junctions”.

[0011] Retroviruses appear to be the best vectors for transferringexogenous genes into eukaryotic cells, especially human cells.

[0012] However, an essential precondition for the use of retrovirusesfor therapeutic purposes and especially in gene therapy is to verify thesafety of their use.

[0013] The principle danger of the use of retroviruses in gene therapyis the possibility of dissemination of a wild-type retrovirusreconstituted in the cell population or tissue considered.

[0014] Such a proliferation could lead to multiple integrations of theretrovirus genome into the genome of the infected cells which can leadto all types of genetic disorders.

[0015] This type of approach therefore poses the problem of reaching thegreatest number of target cells possible, and therefore the capacity ofthis system to reach the greatest number of tumor cells possible, whileensuring the control of virus proliferation.

[0016] Numerous approaches have been established up until now in orderto develop packaging cell lines which produce only defectiveretroviruses carrying the transgene of interest; the development of suchcell lines has considerably increased the use of retroviruses in genetherapy by virtue of the safety conferred by these systems.

[0017] The first vectors used were retroviral vectors carried bypseudo-retroviral particles having an amphotropic envelope in order toallow their use in different species, and defective in order to controltheir dissemination. The initial natural virus is generally the Moloneymurine virus from which the cis-acting elements and the trans-actingelements have been separated in order to form two defective genomes.

[0018] The actual viral vector preserved the essential cis sequences:the LTRs for the control of transcription and integration, the psisequence necessary for encapsidation, the PB sequence necessary for theviral replication. The viral genes (gag, pol, env) are deleted andreplaced by the transgene to be expressed in a target cell placed inprinciple under its own promoter or under a promoter judged to be morepowerful or regulatable.

[0019] The retroviral genes gag, pol and env are often integrated intoanother vector, sometimes called Helper, which is defective for the LTRand POLICE DA_MATH8AwFINP sequences. Their expression allows theencapsidation of the transgene, excluding the genes necessary for themultiplication of the viral genome and for the formation of completeviral particles.

[0020] The proviral form of the Helper is in general integrated into thegenome of a murine cell line (for example fibroblast NIH/3T3) which actsboth as host for the vector and Helper for the functions which it lacks.After transfection of the vector, the cellular strain becomes capable ofproducing defective infectious viral particles. However, these particlescontain only the gene to be transferred (transgene) alone but do notcontain the information necessary for the reconstitution of completeviral particles in the target cells. This system is therefore designedto prevent any subsequent propagation of viruses after the firstinfection, that is to say if the infectious virus carrying the transgenepenetrates into a cell lacking the information of the Helper type (gag,pol and env), its production is stopped.

[0021] Therefore, conventionally, the packaging cell line is a cell linecapable of providing the Helper information in trans relative to theviral genome present in the particle, such that defective infectiousviral particles can be reconstituted.

[0022] Different packaging systems allowing the production of defectiverecombinant viruses have been described in the following documents:

[0023] Patent Application EP 0,243,204 describes the use of retrovirusesas means for packaging any substance in order to cross the membrane ofeukaryotic target cells;

[0024] Patent Application WO 89/07150 describes a retrovirus packagingcell, both plasmids expressing in trans the different viral genes (env,gag, pol), but none of the two comprising the packaging POLICEDA_MATH8AwFINP sequence; the production of virus in these lines isbetween 104 and 106 CFU/ml;

[0025] Patent Applications WO 90/02806, WO 90/12087, EP 0,476,953, WO93/04167 and WO 93/10218 are publications describing gene transfers invivo using retroviral vectors which are themselves produced by packagingcell lines.

[0026] The numerous references of constructs and applications cited inthese patents are included by way of references cited in the presentapplication.

[0027] Recent developments have led to the use of a gene transfer invivo by directly transferring the vector-producing packaging cell linesfor the treatment of tumors; the elimination for example of microscopicexperimental cerebral tumors by stereotactic injection of cellsproducing retroviruses HSV1, followed by treatment with GCV, has beenreported by Culver et al. (Science 256:1550-1552, 1992).

[0028] Likewise, some of the authors of the present application haveshown that, in rats, the in vivo transfer of the HSV1-TK gene by directinjection of murine fibroblasts producing recombinant retroviralparticles, leads to a considerable reduction in hepatic tumors in rats(Caruso M. et al., Proc. Natl. Acad. Sci. USA, 90:7024-7028, 1993 andYves Panis et al., C. R. Acad. SC.

[0029] Paris, Tome 315, Series III, p. 541-544); these articlesdemonstrate, in addition, a very large decrease in the number of cancercells in the tumors, knowing that, surprisingly, the number of cellstransduced in vivo by this type of technique is no doubt less than 10%for TK-HSV1.

[0030] This effect could be explained by the metabolic cooperationmechanism already mentioned above.

[0031] This system of packaging cell lines allowing the production ofviral particles carrying a transgene is therefore extremely promisingfor use in gene therapy and especially for the expression of conditionalgenes.

[0032] These systems, developed and described in the numerous referencescited above, have nevertheless certain limits which are:

[0033] 1) the low productivity of these cell lines (infectious titerless than or equal to 106 PFU/ml), the normal retrovirus Mo MuLVproducing about 108 PFU/ml and a virus such as adenovirus about 109;

[0034] 2) a second limit of this type of technique is that it usesfibroblast cell lines NIH/3T3 which are adhering lines; this is anadvantage when the cell supernatant containing the viral particles isused, but on the other hand is a disadvantage when it is desired to usethe transfected lines themselves as medicinal products, the linesinjected in vivo producing, in this case, the recombinant viralparticles in situ;

[0035] 3) a third disadvantage, which is the other side of the safetyadvantage of the system, is that if the viral particles expressed insitu by the packaging cell line can transfer the transgene of interestinto the dividing cells, any subsequent dissemination is blocked at thislevel. This limits the efficiency of the transfer of the transgenebecause of the blocking of the dissemination.

[0036] It was therefore important to design a system which possiblyallows self-maintenance of the production of recombinant viral particlescarrying the gene of interest while being assured of controlling anysubsequent propagation at the chosen time in order to preserve therequired safety conditions, namely the total control of thedissemination of the virus in its wild-type form.

[0037] In all that follows, the term “transgene” designates the gene tobe expressed in the target cell, which gene, in the examples, is of thesuicide gene type.

[0038] The transgene may also be a cytokine or a molecule of therapeuticinterest.

[0039] The expression “recombinant pseudo-retroviral sequence” meansthat the sequence contains all the genes necessary for the expression ofa pseudo-retroviral particle with the exception of the envelope (env)gene and possibly comprising one or more transgenes.

[0040] The expression “host cell” means the cell containing the tworecombinant nucleic acid sequences, which are carried or not by the sameplasmid and which is capable of being used either as a medicinalproduct, or to produce the defective recombinant viruses.

[0041] The expression “target cell” means the cell which it is desiredto treat by introduction of the transgene.

[0042] The present invention addresses and makes it possible to overcomethe limits of the above-mentioned existing systems by the constructionof a host-vector system which makes it possible to express a transgenein a target cell or a human or animal tissue, characterized in that itconsists of a eukaryotic cell established as a line, into which therehave been transfected:

[0043] a) a recombinant pseudo-retroviral sequence in which the env genehas been deleted totally or partially and substituted by the saidtransgene at the level of the env gene as represented in FIG. 1a, suchthat the transgene is expressed in spliced transcripts similar to thoseof the env gene in the wild-type Moloney virus;

[0044] b) a nucleic acid sequence including a sequence encoding anenvelope or membrane protein, which sequence is in dependence on apromoter and is combined, where appropriate, with the said transgene,and flanked at its 3′ end a polyadenylation site and represented in FIG.1b; the said recombinant viral genome and the said sequence beingcapable of trans-complementing each other and allowing the said hostcell to produce defective infectious viruses lacking the env gene.

[0045] The recombinant viral genome described in a) and the nucleic acidsequence carrying the env gene described in b) may be carried either bytwo different plasmids or transfection systems, either by the sameplasmid or transfection system provided that the b) sequence is situatedoutside the pseudo-retroviral sequence between the two LTRs. A constructon a single plasmid is represented in FIG. 4a.

[0046] The characteristic common to both types of approaches: twoseparate supports or a single support for the a) and b) sequences isthat the b) sequence, being devoid of the ψ sequence, is never packagedand consequently the viruses produced by the host are devoid of it.

[0047] In addition, in the latter case, the plasmid carrying the two a)and b) sequences or that carrying the pseudo-retroviral sequence of theabove FIG. 1a may carry other recombinant sequences intended to enhancethe stability or the duration of expression of the said sequences. Thesecomplementary elements may be for example the ITRs (for: InvertedTerminal Repeats) of the AAV virus (Adeno Associated Virus). An exampleof such a construct is represented in FIG. 4b.

[0048] It appears clearly in this system that the host cell harbouringthe above two recombinant sequences carried by one or two plasmidscannot be likened to a packaging cell line since the first recombinantpseudo-retroviral sequence carries the psi sequence and allows, when itis transfected alone, independently of the second sequence containingthe env gene, the production by the host cell of viral particlescarrying all the initial sequence, but which are devoid of envelope andtherefore noninfectious.

[0049] The cell therefore indeed behaves like the host for a normal orrecombinant viral vector allowing the production of viral particleswithout addition of Helper or trans co-expression as described above.

[0050] The addition to this host cell of a gene encoding the viralenvelope protein in dependence on a promoter and followed by apolyadenylation sequence allows the transcription and then thetranslation of this gene into protein in the host cell and,subsequently, the reconstitution of viral particles carrying the samegenome as the previous ones (and represented in FIG. 1 a) with theexception that they now possess an envelope which allows them to infecttarget cells. These infectious viral particles are, after infection of atarget cell, capable of producing infectious viral particles only if theenv gene is again added and is expressed.

[0051] The recombinant plasmids of the invention which have thecharacteristics of possessing the recombinant retroviral sequencedelimited by the two LTRs, and an env. gene carried or not by the sameplasmid support, may also be combined with a system allowing theirintroduction into the target cells. These systems may be of severaltypes such as viruses or an appropriate vehicle promoting the transferof the genetic material into the cells; the appropriate vehicles aredefined as allowing the crossing of the biological membranes and may beespecially liposomes, cationic lipids, polylysine derivatives,inactivated adenoviruses or ballistic methods.

[0052] Liposomes have been used to enclose and transfer into cells bothnucleic acids and viral particles (R. Philips et al., (1994) Molecularand Cellular Biol. Vol 14 No. 4, p 2411-2418). These vehicles have avery poor efficiency for transfection of the plasmids which theycontain. The addition to the pseudo-retroviral sequence of the inventionof sequences allowing an enhanced and prolonged expression of thetherapeutic sequences of interest makes it possible to envisage theiruse as recombinant plasmid transporters/carriers or as an intermediatemeans for the manufacture of a medicinal product, or as activeingredient of a medicinal product; the cell thus transfected by thevehicle containing the vectors then transforms itself into a cellproducing defective retroviruses encoding the gene of interest butlacking the envelope gene and the defective virus is itself capable ofreinfecting other cells so as to again express therein the gene ofinterest, the cycle necessarily stopping at this stage, unless an env.sequence is added by any possible means. This cell is either the cell ofthe host vector system or the target cell as defined above.

[0053] The invention also relates to the vehicles as defined abovecontaining a pseudo-retroviral sequence as described in 1 a, arecombinant sequence carrying an env. gene as described in 1 b,separated or assembled on the same plasmid support as described in FIGS.4a and 4 b, and optionally carrying ITRs of the AAV virus upstream anddownstream of the recombinant pseudo-retroviral sequence, and preferablydownstream of the recombinant sequence carrying the env. gene.

[0054] More generally, any viral vector in which:

[0055] a gene essential for the constitution of infectious viralparticles is substituted by a transgene of interest,

[0056] this essential gene is present on the same vector, or a separatevector, but is not in dependence on viral promoters and,

[0057] the product of this gene acts in trans and makes it possible toreconstitute defective viral particles, forms part of the invention.

[0058] Another example of a nonretroviral construct of this type is thereconstitution of a defective recombinant adenovirus in which the E1Agene is substituted by a transgene of interest, and the E1A sequence isexpressed in trans so as to reconstitute a defective infectious viralparticle.

[0059] The essential characteristic of the constructs of the inventionis that they make it possible to produce infectious viruses either in ahost cell, or in a target cell, but the viruses produced will contain anucleic acid capable of expressing a transgene and incapable ofproducing infectious particles.

[0060] In the case of the pseudo-viral particles described above, theenv. sequence will be outside the region for regulating the expressionof the retroviral sequences.

[0061] This system is particularly advantageous as will be seen laterwhen the transgene to be expressed is a suicide gene, especially theHerpes Simplex virus thymidine kinase (HSV1-TK), or a cytokine.

[0062] In the host-vector system of the invention, the recombinantpseudo-retroviral sequence is derived from the genome of the Moloneyvirus MuLV, the LTR sequences in 5′ or 3′ being of wild-type origin orderived from different mutants or combinations thereof. There may bementioned for example the LTR type constructs derived from the mutantsmov3, mov9 and mov13 as described in Caruso M. et al. in Proc. Natl.Acad. Sci. USA, 90:7024-7028,1993.

[0063] This nucleic acid sequence (FIG. 1a) which will be encapsuidatedinto the viral particle produced by the host cell, is extremely close tothe “wild-type” retroviral genome which offers the advantage ofobtaining higher titers than those obtained in the systems describedabove in the prior state of the art with the packaging cell lines andthe trans-complementations of the viral genes.

[0064] By way of example, and as will be seen later, whereas in thesystems of the state of the art the maximum titer with the packagingcell lines is of the order of 106, this system allows titers of between106 and 109, and preferably between 107 and 108 PFU/ml.

[0065] A second characteristic of the host-vector system of theinvention is that the host cell is transfected simultaneously with anucleic acid sequence including a sequence encoding an envelope protein,this protein being of viral or even retroviral origin, or a membrane oreven cellular protein.

[0066] “Simultaneously” means that the two sequences, thepseudo-retroviral sequence and the nucleic acid sequence encoding theenvelope gene, are expressed simultaneously in the host cell, it beingpossible to perform the transfections either simultaneously in one ortwo constructs, or successively.

[0067] The envelope gene chosen may be homologous to thepseudo-retroviral sequence, that is to say derived for example fromMo-MuLV, allowing the reconstitution of the homologous pseudo-viralparticles or may be derived from another virus, for example and withoutbeing limiting the vesicular stomatitis virus (VSV), RIV, the rabiesvirus or the gibbon leukemia virus, which gene, when it is transcribedand translated into envelope protein, allows the reconstitution of apseudo-viral type whose envelope is a VSV (or HIV) envelope and thegenome containing the LTRs, psi, PB, gag and pol of MuLV, as well as thetransgene.

[0068] Finally, the env genome may be of cellular origin and may encodea membrane protein allowing the targeting of the viral particle on aspecific ligand, especially for a CD4 type receptor.

[0069] In addition, the envelope may be a chimeric protein whosecarboxy-terminal end is derived from intramembrane sequences of theMoloney envelope. Experimental data indeed exists which shows that thesemembrane sequences may be involved in the concentration of theseenvelope proteins at the surface of the viral particle. With thesechimeric molecules, the efficiency of expression of the chimeric envprotein at the surface of the virions will be increased.

[0070] A second advantage of this host-vector system allowing theexpression of these heterologous pseudo-viral types is the possibility,where appropriate, of purifying these pseudo-viral particles byultracentrifugation without loss of infectious power. This makes itpossible to obtain viral suspensions whose titer may be as high as 109PFU/ml.

[0071] The nucleic acid sequence containing the env gene as described inFIG. 1b below, may be transfected by any type of means, either physicalmeans or with the aid of viral or retroviral vectors. Among the physicalmeans known, there may be mentioned microinjection, liposomes oralternatively so-called biolistic or bombardment processes as describedin Kriegler, M. Gene Transfer and Expression; A Laboratory Manual;MacMillan Publishers Ltd.; 1990.

[0072] The transfection may also take place by integrating this nucleicacid sequence into a viral vector and especially an adenovirus.

[0073] The advantage of the latter method is the possibility of puttingin place a system which is self-maintained in vivo while retaining fullcontrol over the viral proliferation.

[0074] Indeed, the host-vector system comprising, as transgene, asuicide gene and injected for example in a tumor, will express arecombinant viral particle which will itself reinfect in situ dividingcells.

[0075] These cells will in turn reexpress a viral particle, but thelatter will not be infectious since the viral genome will not have beentrans-complemented by an envelope gene.

[0076] If, at this stage, a recombinant expression vector containing theenv gene is injected, it will be possible for a newtrans-complementation to occur and the cycle is repeated once again; thesafety of the system is, in this manner, preserved since a doubletransfection is necessary, one by the pseudo-viral particle MuLV capableof infecting only dividing cells and the other by the adenovirus capableof infecting all the cells. Therefore, only the dividing cells will becapable of being co-transfected, of expressing the viral genes in transand of producing pseudo-viral particles capable of infecting the targetcells.

[0077] This system therefore makes it possible, in a particularlyelegant manner, to transfect into a target cell, for example the TKsuicide gene, allowing the expression of the latter and conferring onthe cell containing it the sensitivity to the nucleoside analogs; thesimultaneous transfection of an envelope gene allows the production, bythis same target cell, of infectious particles comprising the samerecombinant pseudo-viral genome defective in the envelope gene.

[0078] This cycle can therefore be repeated the desired number of timesas long as the sequence carrying the env gene is provided exogenously;the system stops after one replicative cycle of the virus in the targetcell, production of infectious particles, reinfection and production ofnoninfectious particles.

[0079] The whole of this cycle is described in FIG. 2.

[0080] A third advantage of the host-vector system according to theinvention is that any type of eukaryotic cells established as lines maybe chosen provided that this line is capable of producing this type ofamphotropic viral particles.

[0081] In addition to the NIH/3T3 lines conventionally used to producerecombinant viral particles of the Moloney virus, the host-vector systemof the invention allows the use of lines capable of being cultivated insuspension; by way of example, there may be mentioned mouse myelomas,VERO cells or insect cells.

[0082] The obvious advantage of using cells in suspension is theindustrial application for the preparation of medicinal products whichcan be used in gene therapy, large quantities of these cells beingnecessary for such preparations which are obviously much easier toobtain with cells in suspension than with adhering cells.

[0083] The choice of VERO cells as host cells may have the advantage, onthe one hand, of coming from primates and therefore of being morephylogenetically suitable for man and, on the other hand, of being moreresistant to the action of the natural antibodies and of the complement.These cells have finally been used widely for the production ofvaccines, and in particular of viruses which undergo only very fewinactivation or purification stages (for example vaccine againstrabies). Some cells which can be used as viral particle-producing hostcells have the property that the said particles are not inactivated bythe complement: these are especially human cell lines or lines derivedfrom vison.

[0084] The choice, as host cells, of Lepidoptera cells or more generallyof insect cells (with the exception of Diptera) also has many advantagesfor constructing hostvector systems which can be used as medicinalproducts in gene therapy:

[0085] they are capable of producing viral envelope proteins of viruseswhich infect eukaryotes, but they themselves are not capable of beinginfected by these viruses;

[0086] insect cells do not produce mammalian viruses; viral safety testsrelating to them are therefore reduced; these cells are not capable ofbeing infected by viruses which are potentially pathogenic for man; itis therefore safer to use these cells for a use in human therapy fromthe point of view of viral safety tests;

[0087] insect cells grow in suspension and are therefore capable ofbeing cultured on a large scale; insect cells are capable of intratissuemovements; consequently, in the case where they might be injected into atumor, they can therefore move and deliver their pseudo-viral particlesat a distance from the site of injection:

[0088] it is possible to use promoters specific to insect cells whichare strong promoters: for example, it is possible to use baculo viruspromoters which give high expression levels and therefore an increasednumber of pseudo-viral particles; given that there are several strongpromoters derived from baculo virus, it is therefore possible to performcomplex constructions in order to limit the risks of recombinationbetween the different transfected genes.

[0089] Because of all these advantages, insect cells, optionallymodified genetically so as to be capable of manufacturing amphotropicpseudo-viral particles, are a preferred tool for the construction of thehost-vector system of the invention.

[0090] Persons skilled in the art will know, on a case-by-case basis,depending on the transgene which they wish to express in a target cell,how to produce the constructs using the appropriate expression vectorsand the appropriate host cells allowing the trans-complementation of theenvelope gene with the retroviral genome carrying the psi sequence, thegag gene, the pol gene and the transgene(s), and allowing disseminationby the addition of the env gene, which is controlled over time and inspace.

[0091] Another advantageous improvement of the host-vector system of theinvention consists in integrating, into the nucleic acid sequencecarrying the env gene, a sequence of the HSV1 thymidine kinase gene orany other conditional suicide gene; this improvement is a second keywhich makes it possible to increase the efficiency of the treatment, aswell as the safety of the system, since, in this manner, the suicidegene is expressed in the target cell in dependence on promoters ofdifferent types and, consequently, this makes it possible to increasethe sensitivity to nucleoside analogs which is conferred on the cells bythe expression of this suicide gene.

[0092] Another particularly advantageous improvement for the viralsafety problets in a therapeutic use in man is the use of a host-vectorsystem in which:

[0093] the host cells are insect cells, not producing mammalian viruses;

[0094] the vector carrying the env gene used in dependence on a promotercontains upstream of the env gene a homologous sequence of the defectiverecombinant retrovirus used in the host-vector system, for example allor part of a gag gene or all or part of a pol gene.

[0095] This system is advantageous because, if a homologousrecombination with a retrovirus occurs in a system where the hostcontains endogenous retroviruses, for example in mouse cells, then itwill lead to the production of nonfunctional retroviruses. Thisrecombination will occur such that the recombinant genome resultingtherefrom will also be defective. The injection of this type of cellsinto man will be completely safe from the viral point of view.

[0096] Likewise, the use, with this system, of an insect cell in thehost-vector system cannot lead to homologous recombinations, the insectcells not carrying mammalian retroviruses and, consequently, injectionfor therapeutic purposes will make it possible to produce only thedefective recombinant viral particles of the invention, excluding viralparticles derived from a homologous recombination and which could thenbe infectious.

[0097] The present invention also relates to a process for expressing atransgene for gene therapy which consists in using the following steps:

[0098] a) construction of a host-vector system by transfection, into aeukaryotic host cell, on the one hand, of a pseudo-retroviral sequencein which the env gene is deleted totally and replaced with thetransgene, for example at the level of the ATG of the said env gene and,on the other hand, a nucleic acid sequence containing, in its structure,a sequence encoding an envelope protein under the control of a promoter,where appropriate combined with the transgene and flanked at its 3′ endby a polyadenylation sequence, the two sequences above being carried byone or two plasmid type vectors,

[0099] b) bringing the said system into contact with the cells in whichthe transgene has to be expressed,

[0100] c) where appropriate, again transferring, into the target cells,the abovementioned sequence containing the env gene. In the case wherethe two sequences are carried by the same construct, the sequencecontaining the sequence encoding an env. protein is outside the two 5′and 3′ LTRs of the pseudo-retroviral sequence.

[0101] In the process of the invention, the transgene may be a gene oftherapeutic interest and especially a suicide gene such as the HSV1thymidine kinase gene which confers, as stated above, the sensitivity ofthe cells expressing this gene to nucleoside analogs.

[0102] In the process of the invention, the pseudo-retroviral sequenceis directly derived from the Moloney virus MuLV, the LTR sequences in 5′or in 3′ being derived directly from specific viral species such asmov9, mov3 or mov13, they may be of wild-type, mutant or combinedorigin.

[0103] In the process of the invention, the sequence containing the envgene is chosen especially from the retrovirus sequences encoding thesaid gene and especially the env gene of MULV or the env gene of aheterologous virus such as for example VSV, HIV, the rabies virus, theGibbon leukemia virus. It goes without saying that the reconstitutedviral particle is a hybrid particle whose envelope consists of theenvelope protein of the virus from which the env sequence was derivedand the viral genome being derived from MuLV.

[0104] The process according to the invention is characterized in thatthe sequence containing the env gene (whether this sequence is includedin a plasmid also carrying the pseudo-retroviral sequence or on thecontrary whether it is included in an autonomous vector for the latter)is introduced into the target cell by any appropriate means, especiallyby a viral vector, such as an adenovirus, or alternatively by physicalmethods, for example bombardment, fusion of liposomes or microinjection.It goes without saying, as explained above, that the use of anadenovirus allows the implementation of a process with a high yieldwhile preserving total safety since the expression of viral particlescan only be achieved in dividing cells and by trans-complementation ofthe pseudo-viral particle derived from the Moloney virus and of theenvelope gene derived from the adenoviral vector.

[0105] One advantageous variant of the process of the invention,especially when the liposomes are used as transfection means, is theaddition of one or more sequences, outside the two LTRS, whose functionwould be a function of stabilizing the expression of thepseudo-retroviral sequence and especially of the transgene.

[0106] An example of these sequences is the ITR (Invented TerminalRepeat) sequences of the AAV virus. These sequences are capable, whenthey are present on either side of sequences to be expressed, andespecially upstream, of conferring a substantial stabilization of theexpression of the said sequences (Philips et al., supra and Flotte T. T.et al. Am. J. Resp. Cell. Mol. Biol, 1992 7: 349).

[0107] Apart from this type of sequences, any type of sequence capableof increasing expression in terms of the level and/or duration may bechosen by a person skilled in the art and integrated into the vector orplasmid for transfection outside the two type a) and b) nucleosidesequences above.

[0108] More generally, the invention relates to a process which allowsthe expression of a transgene for gene therapy, characterized by thesimultaneous transfection, into target cells, of a retroviral genomehaving the general structure represented in FIG. 1a, which genome hasall the sequences necessary for the expression of a viral particle, butlacks the envelope gene conferring on the said viral particle itsinfectious character and, on the other hand, a nucleic acid sequencecontaining, in its structure, a cellular or virus envelope gene, theco-expression in trans of the two sequences thus transfected allowingthe expression of recombinant viral particles which are infectious butlack the said env gene in their genome.

[0109] This co-expression in trans of the two types of nucleic acidsequences can be achieved either in a host cell therefore allowing theproduction of a host-vector system which can be used as such in genetherapy, or as a simultaneous injection into the target cells to betreated, or finally by encapsulation into a vehicle, for exampleliposomes.

[0110] This expression may be achieved both when the sequences arecarried by two different structures, or by the same plasmid structureprovided that the one carrying the env. gene is outside the two 5′ and3′ LTRs of the pseudo-retroviral sequence.

[0111] The iterative character of the system used is clearly apparent toa person skilled in the art by adding, as required, the second nucleicacid sequence carrying the env gene and capable of being expressed underthe control of a promoter.

[0112] The invention also relates to the use, in gene therapy, of thehost-vector system described above and obtained by simultaneoustransfection, into a eukaryotic cell, of two nucleic acid sequenceswhose expression in trans allows the said eukaryotic cell to produceinfectious viral particles carrying the transgene, it being possible forthis use to be advantageously applied to the expression of a suicidegene in target cells to be destroyed.

[0113] This co-expression is achieved by a double transfection ofsequences described in FIGS. 1a and 1 b, or by transfection of a singlestructure carrying the two types of sequences, 1 a and 1 b, of which anexample is represented in FIG. 4a.

[0114] If this single structure carries, in addition, a sequenceenhancing the expression in terms of level or duration, such as the ITRsequences of the AAV virus, the use is particularly advantageous,especially in the case of the use of liposomes as means of transfection,thus enhancing the efficiency of this technique and allowing the use ofthe liposomes carrying recombinant plasmids as intermediate in themanufacture of a host-vector system which can be used in gene therapy oralso as active ingredient of medicinal products which can be used ingene therapy.

[0115] The AAV ITRs produce their maximum effect when they arepositioned one upstream of the 5′ LTR and the other downstream of thepolyadenylation sequence PA, as represented in FIG. 4b.

[0116] Finally, the invention relates to medicinal products which can beused in gene therapy and which are characterized in that they contain,as active ingredient, eukaryotic cells which have been subjected to adouble transfection with the nucleic acid sequences as described inFIGS. 1a and 1 b; the active ingredient of a medicinal product accordingto the present invention may also consist, on the one hand, ofinfectious recombinant viral particles carrying a viral genome in whichthe env gene has been substituted for example at the level of itsinitiation codon by the tranagene to be expressed and, on the otherhand, by a nucleic acid sequence containing, in its structure, theenvelope gene, in dependence on a promoter and, where appropriate,flanked by a sequence encoding, for example, the thymidine kinase gene.

[0117] The invention likewise relates to medicinal productscharacterized in that they contain, as active ingredient, liposomescontaining recombinant DNA sequences, one of which consists of apseudo-retroviral sequence between the retrovirus 5′ and 3′ LTRs,comprising the gag and pol genes and a gene of therapeutic interest, butlacking the env. gene, and another sequence outside the first,containing an env. gene and a polyadenylation sequence and in dependenceon a promoter and, where appropriate, also flanked by a sequenceencoding a gene of interest; in an advantageous embodiment, themedicinal product consisting of these liposomes carrying recombinantsequences is such that the plasmid carrying them comprises, in addition,sequences which make it possible to enhance the expression of the genesof interest and especially those which are between the two LTRs, thusincreasing the efficacy of the said medicinal product.

[0118] The gene of interest for the medicinal products of the inventionis advantageously the gene for thymidine kinase, which, when it isexpressed, confers on the cell harboring it sensitivity to thenucleotide analogs such as gancyclovir or acyclovir.

[0119] A person skilled in the art will be able to implement all thepossible variants of this type of host-vector system, by routineexperimentation.

[0120] Such variants, or equivalents, form part of the invention asclaimed hereinafter.

[0121] The figures and the examples below will make it possible toillustrate the invention more precisely.

[0122]FIG. 1 represents the construction of the two nucleic acidsequences which are to be transfected simultaneously.

[0123] In this figure:

[0124]FIG. 1a represents the defective recombinant Moloney virus inwhich the grey region represents sequences derived from the Moloneyvirus and the X gene on a dark background represents the transgene;

[0125]FIG. 1b represents a Y gene encoding a viral envelope or amembrane protein flanked in 5′ by a promoter and possibly a retrovirussequence, and flanked in 3′ by a polyadenylation sequence;

[0126]FIG. 1c, for memory, indicates the sequence of the wild-typeMoloney virus.

[0127]FIG. 2 represents the principle of the co-expression in trans ofthe production of defective and infectious viral particles allowing thereintroduction, into a target cell, of the defective recombinantretroviral genome.

[0128] In the first part of the diagram, a vector of the typerepresented in FIG. 1a is introduced into the host cell. (It should benoted that this introduction may be achieved, either by transfection, orby infection with a defective retroviral particle as described at theend of this diagram). The result of this expression of the vectorrepresented in FIG. 1a is the production of viral particles whose genomeis derived from this vector but which do not carry at their surface anenvelope protein and which are therefore noninfectious (as representedin the 2nd part of the diagram).

[0129] A vector of the type represented in FIG. 1b, carrying the envgene, is then expressed by transfection in this cell. Thetrans-complementation then allows the expression, at the surface of theviral particles, of the env protein, these viral particles remainingdevoid of the env gene.

[0130]FIG. 3 represents the therapeutic diagram from the generation ofthe cells producing the infectious viral particles carrying thetransgene up to their injection in situ into the tumor as well as theperpetuation of the infectious cycle by in situ complementation with anenvelope gene. The left column outlines the production of defectiverecombinant viral particles as already represented in FIG. 2, whichcells are injected in vivo into a tumor. The defective recombinant viralparticles produced in situ are then capable of infecting tumor cells;the therapeutic effect of the transgene can then be expressed in thesecells and the transduced tumor cells themselves produce viral particlescarrying the transgene and lacking an envelope. The cycle stops at thislevel, unless a vector according to FIG. 1b is subsequently added.

[0131]FIG. 4a represents a plasmid construct in which thetwo—pseudo-retroviral and env.—sequences are carried by the samestructure.

[0132]FIG. 4b is a variant in which the ITRs of the AAV virus have beenadded upstream of the 5′ LTR and downstream of the polyademylation (PA)sequence.

[0133] The transgene is in both cases the HSV1 thymidine kinase gene.

[0134]FIG. 5 represents a specific pair of vectors used to show theefficiency of the system of the invention, FIG. 5a being the vectorpNP-2 containing the HSV1-TK gene, and the plasmid p CRIP GAC-2containing the Moloney virus env gene, as described in Example 3 below.

EXAMPLE I Construction of a Moloney Virus Carrying a Therapeutic GeneDefective for the Viral Envelope

[0135] The vector used is derived from the retroviral vector pMA 245,which is derived from the Moloney virus MuLV and was constructed fromMuLV fragments cloned from different Mov species (Mov-3,-9, and -13) asestablished by Jaenisch et al., 1981, Cell 24: 519-529.

[0136] The genetic construction can be easily performed by personsskilled in the art. A plasmid carrying an infectious Moloney provirus isused as starting material. Such a plasmid, when it is transfected intoan appropriate cell line (for example mouse NIH-3T3 cell), gives rise toinfectious viral particles carrying the wild-type Moloney genome derivedfrom the transfected genetic construct. In this genome, the geneencoding the envelope is replaced by a gene encoding the protein oftherapeutic interest. This construction is performed such that this geneis synthesized from the same spliced transcripts as the “wild-type”envelope gene. For the sake of efficiency of expression of the gene oftherapeutic interest, and also of efficiency of production of retroviralparticles, the gene of therapeutic interest can even be substituted suchthat its ATG codon is exactly in the position of the ATG of the Moloneyenv gene. The gene of therapeutic interest is more easily derived fromthe complementary DNA and therefore contains no intron and also lacks apolyadenylation sequence. The genetic construction is such that ittherefore preserves the natural splicing of the Moloney virus, such thatit preserves the reading frame of the gag and pol genes and such that itpreserves the integrity of the 3′ LTR of the Moloney virus. The entiregenetic construction is performed according to the rules of the art byenzymatic digestion of the DNA and then ligation of the appropriatefragments; these techniques are described in Maniatis et al., 1989,Molecular Cloning, A Laboratory Manual. If necessary, it uses PCR or anyappropriate amplification technique in order to generate geneticfragments at the ends of which the restriction sites appropriate for thecloning are present.

[0137] If necessary also, the different genetic fragments constitutingthe infectious Moloney provirus can be degraded into shorter fragmentsdistributed over the different plasmids, such that they can be handledmore easily in order to be subsequently reassembled in an appropriatemanner. If necessary, finally, site-directed mutagenesis can be used tointroduce either restriction sites necessary for performing the geneticconstruction, or in order to substitute the different genes at precisepositions. At the end of this genetic construction, a plasmid isavailable which, when it is transfected into a cell (for exampleNIH-3T3), is expressed like a wild-type Moloney virus and thereforeresults in the generation of viral particles whose genome consists ofthe provirus itself. Because of the homology between the infectiousprovirus and the newly constructed defective provirus, the synthesis ofthe different RNAs takes place with an efficiency in optimizedproportions which ensure that the number of viral particles which aremanufactured by the cell is very similar to that of a wild-type virus.This number of viral particles is therefore considerably greater thanthat manufactured by conventional encapsidation lines in which thedifferent Moloney virus structural genes are carried by differentgenetic constructs, and in which the retroviral vector which constitutesthe genome of the viral particle is on a different plasmid and has astructure and a size which are very different from those of thewild-type Moloney. It should be noted that the viral particles thusproduced are not infectious because they lack an envelope. This envelopeprotein may be provided independently in this cell by means of a vectorwhose construction is performed below.

EXAMPLE II Construction of the Nucleic Acid Sequence Carrying the EnvGene

[0138] It is indeed possible to transfect, into this cell, a new simplegenetic construct containing a membrane-expressed gene under the controlof a conventional promoter (for example the SV40 virus promoter, acyto-megalovirus promoter, a promoter of a housekeeping gene such asPGK, or even a promoter specific for a given cell type and which willtherefore offer an additional safety during use). This expression vectoralso comprises polyadenylation sequences obtained from different geneticconstructs (growth hormone, β-globin and SV40 polyadenylation sequencesand the like). The membrane gene may be either an envelope of theMoloney virus itself, or an envelope of another virus which can becaptured by the viral particle and expressed at its surface, or even acell membrane protein which may also be present at the surface of theviral particle. This is the case especially for the CD4 molecule whichhas been shown to be capable of being incorporated by differentretroviruses.

[0139] Another embodiment is the construction of chimeric membraneproteins, such that their intracellular part is derived from theintracellular part of the Moloney virus envelope, and theirextracellular part is a membrane protein allowing better targeting ofthe viral particle. The presence of the intracellular regions of theMoloney envelope probably ensures a better efficiency of expression ofthis chimeric envelope at the surface of the viral particle.

[0140] This second genetic construct is also produced such that itcontains no sequence derived from a Moloney virus other than theenvelope gene itself. More precisely, it cannot contain a sequence whichhas not previously been deleted on the previous genetic construct. Inthis way, the possibility of a recombination between this expressionvector and the defective retrovirus as was described above issubstantially minimized, which recombination could result this time inthe production of an infectious Moloney retrovirus. These geneticconstructions are also performed according to the rules of the art asindicated above.

EXAMPLE III Expression of Viral Particles by Trans-complementation ofthe Two Sequences Constructed in Examples I and II Respectively

[0141] The constructs are used to infect myeloma cells p3X63Ag8 or 3T3TK⁻ cells deficient in thymidine kinase.

[0142] The experiment below comprises two control experiments and twotests:

[0143] batch a): a first control of selection medium, in which the cellsare not transfected,

[0144] batch b): a second control of the efficiency of the transfectionin which the cells are transfected with a plasmid containing a geneencoding β-galactosidase (Lac Z)

[0145] batch c): a test in which the cells are transfected with theplasmid pNP-2 alone but which allows the expression of TK but not thepropagation via the manufacture of defective recombinant retroviralparticles,

[0146] batch d): a test in which the cells are co-transfected with thevector pNP-2 and a plasmid carrying the env gene of the Moloney virusMo-MuLV.

[0147] III.1 Description of the Plasmids

[0148]FIG. 5a represents the plasmid pNP-2 which was deposited at CNCMon Feb. 20, 1995 under No. 1-1541. It contains the Mo-MuLV GAG and POLgenes, with, downstream, an HSV1-TX gene under a mutated env gene ATG.Its total size is 10.38 KB. The sites AF III (at 8.60 Kb) and ECoR1 (at10.21 KB) are represented.

[0149]FIG. 5b represents the second plasmid PCRIP GAG⁻², which is anexample of a construct in which the MoMuLV env gene is integrated into asequence containing the MOMuLV sequences, ψ-gag−, pol+, env+ andfollowed downstream by an SV 40 polyA sequence.

[0150] The figures in bracket indicate the distance in number of basepairs.

[0151] III.2 Transfection

[0152] 3T3 TK cells are either nontransfected (batch a)), or transfectedaccording to the conditions described above (batches b), c) and d)).

[0153] In batch b), the transfection efficiency is revealed using achromogenic substrate; for that, the cells are cultured in the presenceof IPTG (induction of Lac operon) and at the end of 24 h in the presenceof x gal whose color changes to blue when it is cleaved by theβ-galactosidase expressed. The presence of the blue color in the cellstherefore reveals the existence of transfection.

[0154] In batches c) and d), the cells are placed in the presence of HATselection medium either at 24 h, or after 5 days of culture when it isexpected to have a substantially greater number of cells resistant underRAT, taking the cell propagation into account.

[0155] III.3 Results

[0156] 1) Transfection efficiency: under the conditions used, theefficiency of transfection with the plasmid lac Z shows values at around5% of transfected cells.

[0157] 2) Clones obtained after a selection immediately after the 24thhour. For the transfections with the plasmid pNP-2 alone, fewer thanabout ten clones of very small size are seen (between 20 to 50 cells perclone). For the transfection with pNP-2 plus the gene encoding theMoloney envelope, about ten clones of small size plus 38 clones ofconsiderably larger size (>200 cells per clone) are also seen.

[0158] 3) Clones obtained after an RAT selection on the 5th day. For thetransfections with pNP-2 alone, a few clones, also of small size, arefound. For the dish corresponding to the transfection of pNP-2 and theplasmid env, a very large, uncountable, number of clones is observed,corresponding to about a quarter of the confluent state on the dish as awhole.

[0159] III.4 Conclusions

[0160] 1) The plasmid pNP-2 is functional and allows the expression ofthe TK gene.

[0161] 2) Immediately after the 24th hour, recombinant viral particlesare produced which make it possible to efficiently and stably transferthe Tk gene into the cells of the culture.

[0162] 3) After 5 days, a large increase (but difficult to quantify inthis experiment) is observed in the number of stable clones obtained.

EXAMPLE IV Construction of a Single Plasmid Carrying Both theRecombinant Pseudo-retroviral Sequence Lacking the Env. Gene andCarrying the HSV1 Thymidine Kinase, and an Env. Gene in Dependence on aPromoter Upstream and an Env. Gene Downstream

[0163] A conventional plasmid is used as basic skeleton, for example PBR322 by the methods described above. The constructs as represented inFIGS. 4a and 4 b are produced, the plasmid thus recombined may thus beemulsified and enveloped in liposomes or combined with any other vectorwhich makes it possible to cause it to penetrate into the host cell orinto the target cell.

EXAMPLE V Experimental Results Obtained on Established Tumors from theHost-vector System Obtained with the Constructs Described in Examples I,II and III

[0164] It was observed that this host-vector system thus constructedproduces infectious retroviral particles at a titer of 108 particles/ml.

[0165] The titer was checked by infection of mouse L-cells deficient inthymidine kinase and infected according to the method described inCaruso M. et al., Proc. Natl. Acad. Sci. USA, 90:7024-7028, 1993.

[0166] In conclusion, the process for treating cells in vivo by genetherapy with the aid of the host-vector system and the medicinalproducts containing this system as active ingredient appears to beparticularly advantageous in the light of the preexisting techniques onthe different points mentioned above, namely:

[0167] the productivity of the viral particles emitted (at least 1 to 2logs above the productivity of the conventional packaging cell lines),

[0168] the operational nature from the point of view of an industrialimplementation of the production of a medicinal product using thissystem since this host-vector system can use host cells capable of beingcultured in suspension and therefore on a large scale,

[0169] an increase in the efficiency of the system from the point ofview of the target cells since the production of infectious viralparticles carrying the transgene is an iterative process which can berepeated in vivo by addition, with the aid of a vector or of anyappropriate means, of a nucleic acid sequence carrying the env gene andin dependence on its promoter; the system may be blocked which makes itpossible to stop any dissemination of recombinant viral particles bystopping the addition of the env gene; this makes it possible tocontrol, for example in the case of suicide genes, the number of cellsdestroyed by addition of nucleoside analogs according to the size of apossible tumor or the number of cells to be destroyed,

[0170] finally, this system allows great flexibility during use whileretaining viral safety and can be applied to any other conditional geneexpression system.

1. Host-vector system which makes it possible to express a transgene ina target cell or a human or animal tissue, characterized in that itconsists of a eukaryotic cell established as a line, into which therehave been transfected: a) a recombinant pseudo-retroviral sequence inwhich the env gene has been deleted totally or partially and substitutedby the said transgene at the level of the env gene as represented inFIG. 1a; b) a nucleic acid sequence including a sequence encoding anenvelope protein, which sequence is in dependence on a promoter and iscombined, where appropriate, with the said transgene, and flanked at its3′ end a polyadenylation site and represented in FIG. 1b; the saidrecombinant viral genome and the said sequence being capable oftrans-complementing each other and allowing the said host cell toproduce defective infectious viruses lacking the env gene.
 2. Systemaccording to claim 1, characterized in that the sequence in a) and thesequence in b) are carried by two separate plasmids.
 3. System accordingto claim 1, characterized in that the recombinant sequences in a) and inb) are carried by the same plasmid provided that the sequence 1 b) isoutside the region for regulating the expression of the recombinantpseudo-retroviral sequence in a), in particular outside the LTRs. 4.System according to claim 3, characterized in that the plasmid contains,in addition, one or two AAV ITR sequences situated upstream, or upstreamand downstream of the LTRs.
 5. System according to one of claims 1 to 4,characterized in that the nucleic acid sequence including a sequenceencoding an envelope protein comprises, in addition, upstream of thesaid sequence, a homologous sequence of the defective recombinantretrovirus used in the host-vector system, chosen especially from all orpart of the gag gene or all or part of the pol gene.
 6. System accordingto one of claims 1 to 5, characterized in that the transgene to beexpressed is a suicide gene, especially the Herpes Simplex virusthymidine kinase (HSV1-TK).
 7. System according to one of claims 1 to 5,characterized in that the recombinant pseudoviral sequence is derivedfrom the Moloney genome MuLV, the LTR sequences in 5′ or in 3′ being ofwild-type, mutant or combined origin.
 8. System according to one ofclaims 1 to 5, characterized in that the nucleic acid sequence encodingthe env gene is of viral origin and is chosen especially from thesequences encoding the MuLV, VSV, EIV and rabies env gene.
 9. Systemaccording to one of claims 1 to 5, characterized in that the nucleicacid sequence encoding the env gene is of cellular origin and consistsof a sequence encoding a membrane protein, allowing the targeting of theviral particle on a specific ligand, especially a protein encoding aCD4-type receptor.
 10. System according to one of claims 1 to 5,characterized in that the env gene is a chimeric protein in which thecarboxy-terminal end is derived from intramembrane sequences of theMoloney envelope.
 11. System according to one of claims 1 to 10,characterized in that the sequence containing the env gene may beintroduced by infection with a viral vector, especially an adenovirus.12. System according to one of claims 1 to 10, characterized in that therecombinant pseudo-viral sequence and the sequence carrying the env.gene are enveloped in vehicles such as liposomes, and introduced intothe cells by transfection.
 13. System according to claim 12,characterized in that the transfectable cells are either host cells, ortarget cells.
 14. System according to one of claims 1 to 10,characterized in that the eukaryotic cell is a cell established as aline and may be chosen especially from the fibroblast lines such as the3T3 line, or lines capable of being cultured in suspension, such asmouse myeloma cells, or VERO cells.
 15. System according to one ofclaims 1 to 10, characterized in that the cells used are Lepidopteracells.
 16. Process for expressing a transgene for gene therapy whichconsists in using the following steps: a) construction of a host-vectorsystem obtained by transfection, into a eukaryotic host cell, on the onehand, of a pseudo-retroviral sequence in which the env gene is deletedtotally and replaced with the transgene, especially at the level of theATG of the said env gene and, on the other hand, a nucleic acid sequencecontaining, in its structure, a sequence encoding an envelope proteinunder the control of a promoter, where appropriate combined with thetransgene and flanked at its 3′ end by a polyadenylation sequence, b)bringing the said system into contact with the cells in which thetranagene has to be expressed, c) where appropriate, again transferring,into the target cells, the abovementioned sequence containing the envgene.
 17. Process according to claim 16, characterized in that thepseudo-retroviral sequences and the sequence carrying an env. gene areenveloped in vehicles.
 18. Process according to claim 17, characterizedin that the transfection vehicle is chosen by liposomes, cationiclipids, polylysine derivatives, inactivated adenoviruses or a ballisticmeans.
 19. Process according to claim 16 or 17, characterized in thatthe pseudo-retroviral sequence and the sequence carrying the env. geneare carried by the same plasmid.
 20. Process according to claim 19,characterized in that the plasmid contains, in addition, one or two AAVITR sequences situated upsteam, or upstream and downstream of the LTRe.21. Process according to one of claims 16 to 19, characterized in thatthe transgene is a suicide gene, especially the HSV1 thymidine kinasegene, and in that the cells into which the said transgene is integratedare destroyed by addition of a nucleotide analog, especially gancycloviror acyclovir.
 22. Process according to claim 16, characterized in thatthe pseudo-retroviral sequence is directly derived from the Moloneyvirus MuLV, the LTR sequences in 5′ or in 3′ being of wild-type, mutantor combined origin.
 23. Process according to one of claims 16 to 19,characterized in that the sequence encoding the env gene is chosenespecially from the sequences of viruses encoding the said gene,especially MULV, or VSV, RIV, or the rabies virus or sequences encodingcell membrane proteins, especially CD4.
 24. Process according to one ofclaims 16 to 19, characterized in that the sequence containing the envgene is introduced into the target cell by a viral vector, especiallyderived from an adenovirus.
 25. Process according to one of claims 16 to19, characterized in that the sequence containing the env gene isintroduced into the target cells by physical methods, especially bybombardment, fusion of liposomes, microinjection.
 26. Process accordingto one of claims 16 to 19, characterized in that the cell used is chosenfrom established lines not expressing the transgene of interestconsidered, especially mouse 3T3 fibroblast lines, mouse myelomas orinsect cells, or VERO cells.
 27. Process allowing the expression of atransgene for gene therapy which consists in a double transfection ofthe target cells, on the one hand, with defective infectious virusesobtained by the host-vector system of claim 1 and, on the other hand,with a nucleic acid sequence containing in its structure a retrovirusenvelope gene, the in vivo co-expression in trans of the two sequencesallowing the expression of infectious recombinant viral particles, butlacking the said env gene in their genome.
 28. Use, in the manufactureof a medicinal product for gene therapy, of a host-vector systemobtained by the following successive steps: a) simultaneoustransfection, into a eukaryotic host cell, on the one hand, of apseudo-retroviral sequence in which the env gene is deleted totally andreplaced with the transgene and, on the other hand, a nucleic acidsequence containing, in its structure, a sequence encoding an envelopeprotein under the control of a promoter, where appropriate, combinedwith the transgene and flanked at its 3′ end by a polyadenylationsequence, and, where appropriate, combined at its 3′ end with ahomologous sequence of the defective recombinant retrovirus used in thehost-vector system of a gene encoding a retroviral protein, b) bringingthe said system into contact with the cells in which the transgene hasto be expressed, c) where appropriate, again transferring, into thetarget cells, the abovementioned sequence containing the env gene. 29.Use according to claim 28, characterized in that the pseudo-retroviralsequence and the nucleic acid sequence carrying an env. gene are carriedby a single plasmid.
 30. Use according to claim 29, characterized inthat the plasmid contains, in addition, one or two AAV ITR sequencessituated upstream, or upstream and downstream of the LTRs.
 31. Useaccording to claim 28, characterized in that the pseudo-retroviralsequence and the sequence carrying the env. gene are carried by twoseparate plasmids.
 32. Use according to one of claims 27 to 30,characterized in that the gene to be expressed is a suicide gene. 33.Use according to claim 32, characterized in that the suicide gene is thethymidine kinase gene, the target cells in which the gene is expressedbeing destroyed by addition of a nucleoside analog, especially acycloviror gancyclovir.
 34. Medicinal product which can be used in gene therapy,characterized in that it contains, as active ingredient, vehiclescontaining a recombinant retroviral sequence substituted, at the levelof the env. gene, by a transgene of interest, and outside the region forregulating the expression of the retroviral sequences, especiallyoutside the LTRs, a sequence carrying an env. gene in dependence on apromoter.
 35. Medicinal product according to claim 34, characterized inthat the plasmid contains, in addition, one or two AAV ITR sequencessituated upstream, or upstream and downstream of the LTRs.
 36. Medicinalproduct which can be used in gene therapy, characterized in that itcontains, as active ingredient, eukaryotic cells which have beensubjected to a double transfection and represented by the host-vectorsystem according to claim
 1. 37. Recombinant viral vector containing atransgene of interest, which can be used in gene therapy, characterizedin that: a gene essential for the constitution of infectious viralparticles is substituted by a transgene of interest, this essential geneis present on the same vector, or a different vector, but is no longerin dependence on the viral promoters and, the product of this gene actsin trans and makes it possible to reconstitute defective viralparticles.
 38. Vector according to claim 37, characterized in that thevirus is a retrovirus, and the transgene is substituted in the env gene.39. Vector according to claim 37, characterized in that the virus is anadenovirus and the transgene is substituted in the E1A gene.
 40. Use ofa vector according to one of claims 37 to 39, in the manufacture of amedicinal product which can be used in gene therapy.